A quarter century after the discovery of monoclonal antibodies (mAbs) [G. Kohler and C. Milstein, Nature 256:495-497 (1975)], their therapeutic utility is finally being realized. Monoclonal antibodies have now been approved as therapies in transplantation, cancer, infectious disease, cardiovascular disease and inflammation. Many monoclonal antibodies are in late stage clinical trials to treat a broad range of disease indications. As a result, mAbs represent one of the largest classes of drugs currently in development.
The utility of mAbs stems from their specific recognition of a complex target followed by high affinity binding to that target. Because different CH isotypes have different effector functions, it is desirable to tailor the mAb isotype to the desired effector function. For, example, a mAb bearing a constant region with effector functions, e.g., human IgG1, can be used to direct complement dependent cytotoxicity or antibody-dependent cytotoxicity to a target cell. Alternatively, a mAb with a constant region essentially lacking effector function, e.g., human IgG2 or IgG4, can be used to block signal transduction, either by binding to and neutralizing a ligand, or by blocking a receptor binding site.
Many therapeutic applications for monoclonal antibodies require repeated administrations, especially for chronic diseases such as autoimmunity or cancer. Because mice are convenient for immunization and recognize most human antigens as foreign, mAbs against human targets with therapeutic potential have typically been of murine origin. However, murine mAbs have inherent disadvantages as human therapeutics. They require more frequent dosing to maintain a therapeutic level of mAb because of a shorter circulating half-life in humans than human antibodies. More critically, repeated administration of murine immunoglobulin creates the likelihood that the human immune system will recognize the mouse protein as foreign, generating a human anti-mouse antibody (HAMA) response. At best, a HAMA response will result in a rapid clearance of the murine antibody upon repeated administration, rendering the therapeutic useless. More likely is that a HAMA response can cause a severe allergic reaction. This possibility of reduced efficacy and safety has lead to the development of a number of technologies for reducing the immunogenicity of murine mAbs.
In order to reduce the immunogenicity of antibodies generated in mice, various attempts have been made to replace murine protein sequences with human protein sequences in a process now known as humanization. The first humanization attempts utilized molecular biology techniques to construct recombinant antibodies. For example, the complementarity determining regions (CDR) from a mouse antibody specific for a hapten were grafted onto a human antibody framework, effecting a CDR replacement. The new antibody retained the binding specificity conveyed by the CDR sequences. [See P. T. Jones et al. Nature 321: 522-525 (1986)]. The next level of humanization involved combining an entire mouse VH region (HuVnp) with a human constant region such as xcex31. [S. L. Morrison et al., Proc. Natl. Acad. Sci., 81, pp. 6851-6855 (1984)]. Such chimeric antibodies, which still contain greater than 30% xenogeneic sequences, are sometimes only marginally less immunogenic than totally xenogeneic antibodies. [M. Bruggemann et al., J. Exp. Med., 170, pp. 2153-2157 (1989)].
Subsequently, attempts were carried out to introduce human immunoglobulin genes into the mouse, thus creating transgenic mice capable of responding to antigens with antibodies having human sequences. [See Bruggemann et al. Proc. Nat""l. Acad. Sci. USA 86:6709-6713 (1989)]. These attempts were thought to be limited by the amount of DNA which could be stably maintained by available cloning vehicles. As a result, many investigators concentrated on producing mini-loci containing limited numbers of V region genes and having altered spatial distances between genes as compared to the natural or germline configuration. [See U.S. Pat. No. 5,569,825 to Lonberg et al., (1996)]. These studies indicated that producing human sequence antibodies in mice is possible, but serious obstacles remained regarding obtaining sufficient diversity of binding specificities and effector functions (isotypes) from these transgenic animals to meet the growing demand for antibody therapeutics.
In order to provide additional diversity, work has been conducted to add large germline fragments of the human Ig locus into transgenic mammals. For example, a majority of the human V, D, and J region genes arranged with the same spacing found in the unrearranged germline of the human genome and the human Cxcexc and Cxcex4 constant regions was introduced into mice using yeast artificial chromosome (YAC) cloning vectors. [See PCT patent application WO 94/02602 to Kucherlapati et al.]. A 22 kb DNA fragment comprising sequences encoding a human gamma-2 constant region and the upstream sequences required for class-switch recombination was latter appended to the foregoing transgene. In addition, a portion of a human kappa locus comprising Vk, Jk and Ck region genes, also arranged with substantially the same spacing found in the unrearranged germline of the human genome, was introduced into mice using YACS. Gene targeting was used to inactivate the murine IgH and kappa light chain immunoglobulin gene loci and such knockout strains were bred with the above transgenic strains to generate a line of mice having the human V, D, J, Cxcexc, Cxcex4 and Cxcex32 constant regions as well as the human Vk, Jk and Ck region genes all on an inactivated murine immunoglobulin background. [See PCT patent application WO 94/02602 to Kucherlapati et al.; see also Mendez et al., Nature Genetics 15:146-156 (1997)].
Yeast artificial chromosomes as cloning vectors in combination with gene targeting of endogenous loci and breeding of transgenic strains provided one solution to the problem of antibody diversity. Several advantages were obtained by this approach. One advantage was that YACs can be used to transfer hundreds of kilobases of DNA into a host cell. Therefore, use of YAC cloning vehicles allows inclusion of substantial portions of the entire human Ig Heavy and light chain regions into a transgenic animal thus approaching the level of potential diversity available in the human. Another advantage of this approach is that the large number of V genes has been shown to restore full B cell development in mice deficient in murin immunoglobulin production. This ensures tht thers reconstituted mice are provided with the requisite cells for mounting a robust human antibody response to any given immunogen. [See PCT patent application WO 94/02602 to Kucherlapati et al.; L. Green and A. Jakobovits, J. Exp. Med. 188:483-495 (1998)]. A further advantage is that sequences can be deleted or inserted onot the YAC by utilizing high frequency homologous recombination in yeast. This provides for facile engineering of the YAC transgenes.
As mentioned above, there are several strategies that exist for the generation of mammals that produce human antibodies. In particular, there is the xe2x80x9cminilocusxe2x80x9d approach that is typified by work of GenPharm International, Inc. and the Medical Research Council, YAC introduction of large and substantially germline fragments of the Ig loci that is typified by work of Abgenix, Inc. (formerly Cell Genesys), and introduction of entire or substantially entire loci through the use microcell fusion as typified by work of Kirin Beer Kabushiki Kaisha. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described or related to work in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, and 5,814,318 each to Lonberg and Kay, U.S. Pat. No. 5,591,669 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharm International U.S. patent application Ser. No. 07/574,748, filed Aug. 29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279, filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No. 07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16, 1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762, filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No. 08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10, 1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of which are hereby incorporated by reference. See also European Patent No. 0 546 073 B1, International Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, the disclosures of which are hereby incorporated by reference in their entirety. See further Taylor et al. xe2x80x9cA transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.xe2x80x9d Nucleic Acids Research 20:6287-6295 (1992), Chen et al. xe2x80x9cImmunoglobulin gene rearrangement in B-cell deficient mice generated by targeted deletion of the JH locusxe2x80x9d International Immunology 5:647-656 (1993), Tuaillon et al. xe2x80x9cAnalysis of direct and inverted DJH rearrangements in a human Ig heavy chain transgenic minilocusxe2x80x9d J. Immunol. 154:6453-6465 (1995), Choi et al. xe2x80x9cTransgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosomexe2x80x9d Nature Genetics 4:117-123 (1993), Lonberg et al. xe2x80x9cAntigen-specific human antibodies from mice comprising four distinct genetic modifications.xe2x80x9d Nature 368:856-859 (1994), Taylor et al. xe2x80x9cHuman immunoglobulin transgenes undergo rearrangement, somatic mutation and class switching in mice that lack endogenous IgM.xe2x80x9d International Immunology 6:579-591 (1994), Tuaillon et al. xe2x80x9cAnalysis of direct and inverted DJH rearrangements in a human Ig heavy chain transgenic minilocusxe2x80x9d J. Immunol. 154:6453-6465 (1995), and Fishwild et al. xe2x80x9cHigh-avidity human IgG monoclonal antibodies from a novel strain of minilocus transgenic mice.xe2x80x9d Nature Biotech. 14:845-851 (1996), the disclosures of which are hereby incorporated by reference in their entirety.
In connection with YAC introduction, Green et al. Nature Genetics 7:13-21 (1994) describes the generation of YACs containing 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus, respectively, which contained core variable and constant region sequences. Id. The work of Green et al. was recently extended to the introduction of greater than approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and kappa light chain loci, respectively, to produce XenoMouse(trademark) mice. See Mendez et al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), and U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996, the disclosures of which are hereby incorporated by reference. Such approach is further discussed and delineated in U.S. patent application Ser. No. 07/466,008, filed Jan. 12, 1990, Ser. No. 07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297, filed Jul. 24, 1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filed Ser. No. 08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848, filed Aug. 27, 1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279, filed Jan. 20, 1995, Ser. No. 08/430,938, Apr. 27, 1995, Ser. No. 08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5, 1995, Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837, filed Jun. 5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No. 08/486,857, filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995, Ser. No. 08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct. 2, 1996, and Ser. No. 08/759,620, filed Dec. 3, 1996. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998). See also European Patent No., EP 0 463 151 B1, grant published Jun. 12, 1996, International Patent Application No., WO 94/02602, published Feb. 3, 1994, International Patent Application No., WO 96/34096, published Oct. 31, 1996, and WO 98/24893, published Jun. 11, 1998. The disclosures of each of the above-cited patents, applications, and references are hereby incorporated by reference in their entirety.
In connection with the microcell fusion approach, portions or whole human chromosomes can be introduced into mice as described in European Patent Application No. EP 0 843 961 A1, the disclosure of which is hereby incorporated by reference. It will be understood that mice generated using this approach and containing the human Ig heavy chain locus will generally possess more than one, and potentially all, of the human constant region genes. Such mice will produce, therefore, antibodies that bind to particular antigens having a number of different constant regions. Thus, there is no way to preselect the desired constant region for particular effector function.
Technology exists for in vitro isotype switching of antibodies. Antibodies produced from transgenic mice that produce only IgG1 isotypes, from transgenic mice that produce multiple IgG isotype, or from phage display technologies may have the desired antigen-specificity and affinity, but not have the desired effector function. In this instance, the variable region of the heavy chain, at the least, and most likely, the entire light chain of the antibody must be cloned.
Methods for cloning include recovery of genomic DNA from a library, recovery of cDNA from a library, recovery of genomic DNA using specific oligonucleotide primers, and PCR using specific oligonucleotide primers and cDNA as template (RT-PCR). Each method, especially PCR-based methods, require that clone be sequenced to verify faithful reproduction of the antibody coding sequences. Then the variable region of the heavy chain must be operably linked via DNA ligation to the desired constant region gene. Then, the engineered VH-CH gene must be operable linked to expression controlling regions such as a promoter-enhancer and a polyadenylation site. Such an expression construct might also be needed for the Ig light chain of the antibody.
The expression construct(s) must be stably transfected into a suitable host cell for transcription and translation to produce a secreted form of the engineered mAb. Typically, at the least, extensive screening must be performed to find a clone of the cell line that expresses sufficient levels of mAb for further experiments and subsequent manufacturing. More likely, methodologies such as DNA amplification must be employed to raised the copy number of the antibodies expression constructs and consequently, the expression level of the mAb.
Finally, the re-engineered mAb must be re-tested to confirm that it has retained the desired qualities and has the desire function, including specificity, affinity, and presence or absence of effector function. Other technologies for isotype switching exist, but all such programs to re-engineer the mAb isotype requires experimentation and expertise in molecular biology and tissue culture, and is labor intensive, slow, expensive, and covered by issued and pending intellectual property, requiring additional licensing fees, if even available for licensing. Thus, re-engineering of mAb from one isotype to another requires expertise, extra monetary expenditure and slows down the development of the monoclonal antibody for pre-clinical and clinical trials.
Having a technology that would produce the mAb with the desired Cg isotype a priori would obviate the need for antibody re-engineering. By having three different XenoMouse strains, one each capable of making only Cg2, Cg4 or Cg1, a transgenic mouse can be pulled off the shelf, and then can be immunized to produce mAbs with the desired affinity, antigen-specificity and the desired isotype and with the desired effector function a priori. This increases the efficiency and user-friendliness for development of monoclonal antibody based therapeutics. No expertise in molecular biology or antibody engineering is required. The antigen-specific mAb can be taken directly into pre-clinical studies without the extra expenditure of money and time, resulting in a decrease in the development cost and an acceleration of the timeline for development of the therapeutic mAb.
The present invention is directed to solving the problem of obtaining a pre-selected human antibody isotype from a transgenic mouse, in addition to the desired specificity, which is compatible with the therapeutic goals for which the antibody will be used.
The present invention solves the problems referred to above by providing, in one aspect of the invention, transgenic non-human animals capable of producing high affinity, fully human antibodies of a desired isotype in response to immunization with any virtually any desired antigen. The aforementioned transgenic non-human animals have in their somatic and germline cells an unrearranged human immunoglobulin heavy chain transgene that encodes, on rearrangement, a fully human immunoglobulin heavy chain of the desired isotype. The human immunoglobulin heavy chain transgene in the foregoing animals comprises a human constant region gene segment comprising exons encoding the desired heavy chain isotype, operably linked to switch segments from a constant region of a different heavy chain isotype, i.e., a non-cognate switch region.
The foregoing transgenic non-human animal also has in its somatic and germ cells a human immunoglobulin light chain transgene. In a preferred embodiment, the endogenous immunoglobulin heavy and light chain loci of the transgenic non-human animal are inactivated so that the animal is incapable of producing endogenous heavy or light chains. In a particularly preferred embodiment, the non-human transgenic animal is a mouse.
In another aspect, the invention provides an unrearranged human immunoglobulin heavy chain transgene that encodes, on rearrangement, for a human heavy chain of a desired isotype. The transgenes of the invention comprise a DNA sequence identical to the DNA sequence of human chromosome 14 starting at least from the first D segment gene of the human immunoglobulin heavy chain locus, continuing through the J segment genes and the constant region genes through Cxcexc of that locus. In the transgenes of the invention, the aforementioned DNA fragment is operably linked to and is capable of isotype switching to an additional constant region segment. Said additional constant region segment comprises a switch region and human constant region coding segment, wherein the constant region coding segment is operably linked to a switch region that it is not normally associated with, i.e., a non-cognate switch region. In transgenes of the invention, the foregoing DNA fragment and constant region segment is operably linked to at least one human V segment gene. In one embodiment of the invention, the transgene is a yeast artificial chromosome (YAC).
In the transgenes of the invention, the non-cognate switch region may be a switch region from a different species than the constant region coding segment. In one embodiment, the non-cognate switch region is a mouse switch region operably linked to a human constant region coding segment encoding a human gamma, alpha or epsilon constant region. In a preferred embodiment, the switch region is a mouse gamma-1 switch region. In more preferred embodiments, the switch region is a mouse gamma-1 switch region and the human constant region coding segment encodes a gamma-1 or a gamma-4 constant region. In a particularly preferred embodiment, the transgene is the yH2Bm yeast artificial chromosome (YAC) or the yH2Cm YAC.
In another embodiment, both the non-cognate switch region and the constant region coding segment are human sequences, the non-cognate switch region being from a human constant region of a different isotype than the constant region coding segment. In a preferred embodiment, the switch region is a human gamma-2 switch region and the constant region coding segment is an isotype other than gamma-2. In a more preferred embodiment, a transgene of the invention comprises a human gamma-2 switch region and a human gamma-1 or human gamma-4 constant region coding segment. In particularly preferred embodiments, the transgene is the yHG1 YAC or the yHG4 YAC.
In still another embodiment, a transgene of the invention comprises a human non-cognate switch region and a human constant region coding segment, wherein the switch region and the membrane exons of the constant region coding segment are from the same human constant region isotype and the secreted constant region exons are from a different isotype. In a preferred embodiment, the switch region and membrane exons are from a human gamma-2 constant region. In particularly preferred embodiments, the switch region and membrane exons are from a human gamma-2 constant region and the secreted constant region exons are from a human gamma-1 or a human gamma-4 constant region. In preferred embodiments, the transgene is the yHG1/2 YAC or the yHG4/2 YAC.
In another embodiment, any of the foregoing transgenes of the invention comprise a plurality of different human VH genes. In a preferred embodiment, the transgene comprises at least 50% of the human germline VH genes. In another embodiment, the transgene comprises at least 40 different human VH genes. Preferably, the transgene comprises at least 66 different human VH genes. Most preferably, the transgene comprises the entire human VH region of a human heavy chain locus. In another embodiment, the transgene comprises a sufficient number of different human VH genes so that the transgene is capable of encoding at least 1xc3x97105 different functional human immunoglobulin heavy chain sequence combinations, without taking into account junctional diversity or somatic mutation events. In still another embodiment, the number of human VH genes in the transgene is sufficient to produce at least 50% of the B-cell population of a wild-type mouse in a transgenic mouse containing the transgene.
A transgene of the invention further comprises a murine 3xe2x80x2 enhancer, positioned 3xe2x80x2 of the constant region gene containing the non-cognate switch region. In one embodiment the murine 3xe2x80x2 enhancer is an approximately 0.9 kb core region of the native enhancer. In an alternative embodiment, the 3xe2x80x2 enhancer is an approximately 4 kb region of the murine enhancer that includes the core region. In still another embodiment, the transgene includes the mouse major enhancer locus.
In another aspect, the invention provides methods for producing the transgenic non-human animals of the invention. According to the methods, an unrearranged human immunoglobulin heavy chain transgene is introduced into the germline of a non-human animal to produce a transgenic non-human animal having the transgene in its somatic and germ cells. Breeding of the human heavy chain transgenic animals with transgenic non-human animals containing a human immunoglobulin light chain transgene produces transgenic non-human animals containing a human heavy chain transgene of the invention and a human light chain transgene. Either of the aforementioned transgenic non-human animals can be bred with animals having inactivated heavy and/or light chain loci to produce a transgenic non-human animal that produces a fully human antibody and is incapable of producing an endogenous antibody.
In one embodiment, a transgene of the invention is introduced into an embryonic stem (ES) cell which is then inserted into a blastocyst. The blastocyst with the ES cell containing the transgene of the invention is then surgically inserted into the uterus of the non-human animal to produce a chimeric non-human animal. The chimeric animal is bred to obtain germline transmission of the transgene of the invention to produce a transgenic, non-human animal having somatic and germ cells containing the transgene of the invention. Accordingly, a further aspects of the invention are an ES cell comprising a transgene of the invention and non-human animals having the transgene in some or all of its cells.
In still another aspect, the invention provides a method for producing high affinity, fully human antibodies of a desired isotype that are specific for an antigen of interest in a transgenic non-human animal of the invention. According to the method, a transgenic non-human animal of the invention is contacted with an antigen of interest under conditions that induce the production of an antibody by the B-cells of the animal. High affinity, fully human, antigen-specific antibodies of the desired isotype can be collected from the blood stream of the transgenic non-human animal.
Alternatively, according to the methods of the invention, the antibody producing B-cells can be harvested from the animal and immortalized by any means known in the art, for the continuous production of antibodies. In one embodiment, the B-cells are fused with a mouse myeloma cell-line to produce antibody-secreting hybridomas. Such hybridomas can be screened to select those secreting high affinity, fully human, antigen-specific antibodies.
In a further aspect, the invention provides hybridomas derived from antibody producing B-cells harvested from a transgenic animal of the invention.